Chromatography Forum

Hi,

i was going to replace top frit on stainless steel column and when I opened column, I saw a bubble staring right at me. I run in the column in a Waters HPLC system. Does anyone have any suggestions on how to get rid of bubble? I had been trying to run 20% methanol in reverse flow direction but got such high pressure readings (now I know why). I am so tempted to get a safety pin and pop it but I don't think that will work. Someone told me to scoop it out and add top off gel.
Any advice is welcome. It is a G4000SW silica gel column from TOSOH.

S

This void at the top of the column is caused by either settling of the column packing or dissolution of the silica. Contamination leading to excess pressure can cause the settling. Flushing and cleaning won't help. The suggestion to scoop it out and replace the packing material is the only hope for now. For the future, you may want to invest in a guard column and/or precolumn filter and change them regularly.

Hi,

Thank you for your reply. I did have a guard column and precolumn filter in place, but not during cleaning step with 20% methanol. Should I have had the guard column in place?

S

there isn't a void at the top, it is a bubble.

S

I'm having a hard time imagining what you mean by a bubble, as opposed to a void. Can you describe it in very literal terms? (or a photograph?)

it moves around. like a bubble would in soln, so i know it's a bubble

s

When you take off the end fitting, the column packing should come up exactly even with the end of the column tube, like the right-hand column in this image:


If you have room for a bubble, then you have a void space at the column inlet.

Hmmm...I see. Thanks for the visual.

I ended up popping the bubble with a fine syringe and then twisted on the new end fitting/frit I bought. I set the column in reverse flow direction and ran 20% methanol thru at 0.2ml/min...it was doing fine and then went over max pressure. So, I opened up fitting on one end to see if there was a bubble, closed it up, and then open up the fitting on the other end to see if there was a bubble, and closed it up. I saw no bubbles and the resin was face with the fitting as you showed in your pic. One thing though, one of the resin sides didn't look as smooth, neat. I did order more resin top off and it should be here tomorrow. I may just take out the "choppy-looking" resin and replace.

So, the main problem I am having now is that the pressure keeps going so high when attempting to run methanol to clean column. I have a new guard column and precolumn filter with new frit in place. The column is relatively new, installed early Feb., however, it was used quite a bit in Feb.

Any suggestions?

S

First off, check the pressure without the column (probably not the problem, but best to be sure you don't have a blockage somewhere else in the system).

The most likely explanation is that you have some sort of garbage precipitated on the column packing. The guard cartridge is supposed to catch the crud, but if you go too long before changing the guard, you can get breakthrouth (ask me how I learned that! ). Backflushing with a good solvent for the crud can (usually, but not always) clean it off. You know what kind of samples you've been running, so check if 20% methanol would be a good solvent etc.

Digging out the cruddy packing and refilling can help. It's a PITA, and I find it hard to justify except in a case like yours where you have a very expensive column.

What does PITA stand for?

Yeah, I had used an old guard column and just changed it when I started having such problems.

I have been using a high sodium PBS as an eluent and various MW dextran (labelled with FItC). If you know of a good solvent beside methanol, let me know.

Thanks for your help!
S

oh, i get it "pain in the a**"

lol

A bubble which survives the writing of a forum chain, etc? Are you injecting dextran syrup, laced with honey and glue?
What do you mean by "resin" to fill the gap? G4000SW material? How do you get at that?

can a void cause pressure issues? i really can't tell anymore if i have a void b/c i popped the bubble and everything seems pretty flush, although things are a little uneven. Should I just scoop out resin?

S

It is the other way around. The pressure problem causes the void.

did resin collapse under pressure and that's why there is a void? should i be concerned that rest of column is not ok, i.e., will filling in top with more resin really save the column?